Abstract In the last 15-20 years, tissue repair researchers identified numerous stem cells that restore injured organs. Currently, little is known about how these stem cells are influenced by signals from other organs. This inter-organ communication is especially significant following injury of organs sharing a physical boundary, where repairing tissue must be balanced with maintaining cell identity at the organ-organ boundary. We pioneered study of a simple tissue model of organ boundary repair- the Drosophila midgut-hindgut boundary. In doing so, we not only uncovered a unique stem cell that responds to organ-organ boundary injury, but also discovered a role for ploidy growth by differentiated (non-stem) cells in the same tissue repair process. The biology of these two coordinated organ boundary repair responses is the subject of this proposal. We will reveal regulation of two relatively unexplored aspects of tissue repair with intriguing parallels to injured mammalian tissue. Specifically, injury- induced mis-regulation of stem cells/cell identity at a human organ boundary is linked to gastrointestinal cancer. In addition, differentiated cells repair the liver and several other organs by ploidy-driven growth. We have found that injury repair in our system involves: 1) suppressed division in favor of ploidy/cell size increases in differentiated organ boundary cells and 2) active division of a distinct stem cell at the midgut-hindgut boundary. We argue these two mechanisms cooperate to restore lost tissue mass while maintaining the midgut-hindgut boundary. Using our model system, we can target acute injury to the hindgut epithelium. When we do this, we find differentiated adult hindgut cells near the midgut border do not divide but instead increase in cell size and genome content, a conserved response known as hypertrophy. In AIM1, we will determine why hypertrophy is the primary repair mode in the adult hindgut. To answer this question, we will identify important differences between hypertrophic repair in the adult hindgut and canonical mitosis-based repair in the juvenile hindgut. Expanding on preliminary data, we will explore how specific signaling pathways and transcriptional changes that we have identified distinguish between hypertrophic and mitotic responses. Following the same injury that induces adult hindgut hypertrophy, we find cell division also occurs, but only in a specific population of hindgut- adjacent midgut stem cells. In AIM2, we will precisely define the molecular regulation/cellular output of these organ boundary stem cells. Specifically, we will trace the lineage contribution of these distinctive stem cells and determine the role of signals we have identified in inter-organ repair. We also will examine the function of dynamic transcriptome changes that occur following organ boundary injury. Given that few tissue repair researchers study inter-organ communication or the mechanisms that discern between hypertrophy and division, our work promises significant conceptual advances in understanding tissue repair strategies.